The present invention relates to a method of analyzing polymer using laser ablation and system therefor, more particularly to the method of analyzing polymer using laser ablation and system therefor, which are capable of significantly improving the efficiency of analysis comparing with a conventional ones, and the invention relates to the method of analyzing polymer using laser ablation and system therefor, which are preferably used in mass analysis of various kinds of polymer such as DNA, protein, RNA, PNA, lipid, sugar and the like, for example.
In recent years, the application range of the mass analysis has rapidly spread from the field of physics and chemistry to the field of life science such as medical science and biochemistry. Particularly, its development in decision calculus of protein molecular weight and decision calculus of amino-acid sequence is astonishing.
The principle of such mass analysis is that a sample is ionized by various kinds of method, ions obtained by ionization are separated according to mass/charge, and the intensity of each separated ion is measured.
Incidentally, conventional mass analysis for polymer has been the one that electron was added to the polymer itself to ionize it, its mass was analyzed or a molecule of high molecular weight was fragmented into molecular ions of low molecular weight to perform mass analysis, and constituent molecules were compared.
Herein, as an ion generating method in the conventional mass analysis of polymer, a secondary ion mass spectrometry (SIMS) where high-energy atomic ions are made to collide with polymer to cause ionization, an electron desorption ionization (ED) where the molecule is fragmented into the molecular ions of low molecular weight by electron impact, a matrix-assisted laser desorption ionization (MALDI), and the like are known, for example.
However, all of the above-described methods have had problems that they needed a mass spectrograph having high resolving power to perform mass analysis to polymeric ion and that the existence of fragment ions, which were decomposed/generated halfway, made the analysis of mass spectrum difficult.
On the other hand, as mass analysis method for a polymer sample labeled by isotope in performing chemical analysis, a laser atomization resonance ionization microprobe (LARIMP) where a nano-second laser performs atomization and ionization has been conventionally known.
However, according to the LARIMP method, two lasers that are an atomization laser to atomize a labeled element and a resonance ionization laser to ionize the atoms of the atomized labeled element are required as the laser, there has been a problem that the system configuration became complicate.
Moreover, resonance ionization needs to be performed to the labeled atoms in the LARIMP method as described above. For this reason, it is necessary to irradiate laser beams having a unique wavelength to each labeled atom, and there has been a problem that it was quite difficult to perform efficient analysis in the case where various kinds of labeled isotope were mixed.
The present invention has been invented in consideration of the various kinds of above-described problems that the prior art has, and its object is to provide the method of analyzing polymer using laser ablation and system therefor, which generate the atomic ions of constituent atoms that constitute polymer and analyze the generated atomic ions, and the mass analysis method for polymer using laser ablation and system therefor, which do not require a spectrograph of high resolving power. More specifically, the object is to provide the mass analysis method for polymer using laser ablation and system therefor, which eliminate a chance where the analysis of mass spectrum becomes difficult and where the mass spectrograph does not require high resolving power, in the case of performing mass analysis, for example.
Further, the object of the present invention is to provide the method of analyzing polymer using laser ablation and system therefor, which enables single laser to simultaneously realize atomization and ionization of the constituent atoms that constitute polymer, and to drastically simplify the system configuration.
Furthermore, the object of the present invention is to provide the method of analyzing polymer using laser ablation and system therefor, which are capable of performing efficient analysis even in the state where various kinds of labeled isotope are mixed.
To achieve the above-described objects, the present invention is one that the ultra-short pulse laser beams perform ablation to various kinds of polymers such as DNA, protein, RNA, PNA, lipid, sugar and the like, for example, the polymers are transferred into atomic ions to generate atomic ions, and the generated atomic ions are analyzed. With this configuration, the chemical analysis for various kinds of polymers can be performed.
Specifically, according to the present invention, by performing laser ablation to polymer by the ultra-short pulse laser beams, the polymer is decomposed in pieces and atomized by each of atoms that constitute the polymer, and the atomized atoms are ionized into univalent ions, and quantitative analysis can be performed by analyzing the atomic ions generated by the ionization.
Therefore, when performing mass analysis in the present invention, mass analysis is performed to the atomic ions of low mass, which eliminates the chance where the analysis of mass spectrum becomes difficult. The mass spectrograph does not require high resolving power.
Further, as described above, according to the present invention, by performing ablation to polymer by the ultra-short pulse laser, the ionization of atomized atoms into univalent ions can be efficiently performed simultaneously with the atomization of polymer. Therefore, the system configuration can be simplified, various kinds of labeled elements can be simultaneously used in performing chemical analysis, and thus analysis efficiency can be improved.
In the present invention, since the single ultra-short pulse laser can simultaneously atomize and ionize labeled elements, the system configuration can be drastically simplified.
Moreover, since the above-described ionization is ionization (non-resonance ionization) performed by high peak power intensity of the ultra-short pulse laser beams via non-resonant process, each labeled atom can be ionized even in the state where various kinds of labeled isotopes are mixed, it can be easily applied to a multi-label system, and highly accurate and highly efficient polymeric analysis can be performed.
Thus, the present invention is extremely preferable for the use in the quantitative analysis for gene expression, which will be increasingly important in future.
Specifically, the present invention is the method of analyzing polymer using laser ablation, where polymer is atomized into constituent elements by irradiating laser beams on the polymer, which is the analyzing subject, to perform ablation to the polymer, the atomized constituent elements are ionized, and the ionized constituent elements are thus analyzed, wherein the laser beams irradiated on the polymer, which is the analyzing subject, to perform ablation to the polymer are the ultra-short pulse laser beams, in which the polymer is simultaneously atomized into constituent elements and ionized by irradiating the ultra-short pulse laser beams on the polymer, which is the analyzing subject, to perform ablation to the polymer, and the ionized constituent elements are analyzed.
Herein, mass analysis can be cited as the above-described analysis, for example, and chemical analysis (so-called regular chemical analysis) or optical analysis (such as a fluorescence method), for example, is cited as analysis other than the mass analysis.
Further, in the present invention, the polymer that is the analyzing subject may be one transformed into solid phase (dry phase).
Furthermore, in the present invention, a method of transforming polymer into solid phase may be a method that includes a process of transforming the polymer into solid phase by dropping solution of the polymer, which is the analyzing subject, onto a substrate to dry.
Still further, in the present invention, the above-described substrate is a solid and the thermal conductivity of the solid may be 0.1 W.mxe2x88x921.Kxe2x88x921 or more
In addition, in the present invention, the polymer that is the analyzing subject may be the one added with an elemental label.
Further, in the present invention, the above-described elemental label may be a group 1 element in the periodic table.
Further, in the present invention, the above-described elemental label may be a group 16 element in the periodic table.
Further, in the present invention, the above-described elemental label may be a group 17 element in the periodic table.
Further, in the present invention, the above-described elemental label may be a transition metal element in the periodic table.
Further, in the present invention, the above-described element label may be a stable isotopic label.
Furthermore, in the present invention, the ultra-short pulse laser beams irradiated on the polymer, which is the analyzing subject, to perform ablation to the polymer may have a pulse duration of 10 pico seconds or less and a peak power of 10 megawatt or more.
Furthermore, in the present invention, the ultra-short pulse laser beams irradiated on the polymer, which is the analyzing subject, to perform ablation to the polymer may have the pulse duration of 1 femto second or more and 1 pico second or less, and the peak power of 1 gigawatt or more and 10 gigawatt or less.
Further, in the present invention, the analysis of the above-described ionized constituent element may be mass analysis.
Further, in the present invention, the mass analysis may be mass analysis by a time-of-flight method.
Furthermore, in the present invention, analysis may be simultaneously performed to a plurality of ionized constituent elements.
Still further, in the present invention, the polymer that is the analyzing subject may be nucleic acid or the analog of nucleic acid, which is fixed on a DNA microarray.
It is to be noted that DNA, RNA and PNA, for example, are specifically cited as the nucleic acid or the analog of nucleic acid.
Further, in the present invention, the above-described DNA microarray may be a multi-channel DNA microarray.
Further, in the present invention, by moving at least either one of the short pulse laser beams that perform ablation to polymer and the polymer that is the analyzing subject, the short pulse laser beams that perform ablation to the polymer may perform ablation to the polymer, which is the analyzing subject, without omission and duplication.
Furthermore, the present invention is an analysis system for polymer using laser ablation, where polymer is atomized into constituent elements by irradiating laser beams on the polymer, which is the analyzing subject, to perform ablation to the polymer, the atomized constituent elements are thus ionized, and the ionized constituent elements are analyzed, in which the system has a vacuum chamber capable of arranging a target inside thereof, a spectrograph arranged in the vacuum chamber, and an ultra-short pulse laser that emits ultra-short pulse laser beams to irradiate the target arranged in the above-described vacuum chamber.
Further, in the present invention, the system may further have moving means that moves the target in the above-described vacuum chamber.
Further, in the present invention, the moving means that moves the above-described target may be rotational means that rotates the target.
Moreover, in the present invention, the system may further have moving means that moves the irradiation position of the ultra-short pulse laser beams to the target.
Further, in the present invention, the above-described spectrograph may be a mass spectrograph.
Further, in the present invention, the above-described mass spectrograph may be a quadrupole mass spectrograph.
Still further, in the present invention, the above-described mass spectrograph may be a time-of-flight mass spectrograph.
Further, in the present invention, the above-described mass spectrograph may be a Fourier transform mass spectrograph of ion cyclotron type.
Furthermore, in the present invention, the above-described ultra-short pulse laser may irradiate short pulse laser beams having a pulse duration of 10 pico seconds or less and a peak power of 10 megawatt or more.
Still further, in the present invention, the above-described ultra-short pulse laser may irradiate short pulse laser beams having the pulse duration of 1 femto second or more and 1 pico second or less and the peak power of 1 gigawatt or more and 10 gigawatt or less.
Herein, in performing ablation to polymer by ultra-short pulse laser beams in the present invention, irradiating one shot (one pulse) of ultra-short pulse laser beams to polymer is enough. However, plural shots (plural pulses) of ultra-short pulse laser beams may be irradiated on polymer, and the shot number (pulse number) of ultra-short pulse laser beams irradiated on polymer may be appropriately selected.
In addition, it is preferable that the ultra-short pulse laser has the pulse duration of 10 pico seconds or less, and particularly, it is adequate to use laser of 1 femto second or more and 1 pico second or less, which is regularly referred to as femto second laser. As its peak power, 10 megawatt or more is preferable, and more specifically, 1 gigawatt or more and 10 gigawatt or less is preferable.
This is because multivalent ions are generated to make the analysis of mass spectrum difficult if the output is larger than the above-described range, and the efficiency of atomization/ionization reduces and it becomes impossible to observe an atomic ion signal if the output is smaller than the above-described range.
It is to be noted that, according to an experiment conducted by the inventor, which is described later, it was possible to obtain an excellent result in the case of the pulse duration of 110 femto seconds and the peak power of 2 gigawatt, for example.
Furthermore, according to the present invention, ultra-short pulse laser beams such as the femto second laser beams capable of efficiently performing atomization and ionization simultaneously is made to irradiate on a polymer sample that is labeled by isotope. For this reason, it is not necessary to selectively ionize the labeled elements and various kinds of labeled element can be used. Moreover, since the repetition rate of laser irradiation can be raised to a few kHz, the invention is suitable for high-speed analysis.
Further, in the present invention, by moving at least either one of the short pulse laser beams that perform ablation to polymer and the polymer that is the analyzing subject, the short pulse laser beams that perform ablation to the polymer perform ablation and analysis to the polymer, which is the analyzing subject, without omission and duplication. Specifically, in the present invention, by moving the spot of short pulse laser beams and the substrate on which polymer as the sample, which is the analyzing subject, is coated, ablation to the large number of samples coated across a wide area can be performed without omission/duplication. This is particularly effective in application to the DNA microarray.
In the present invention, due to the above-described characteristics, not only analysis speed becomes remarkably faster than the conventional one but also simultaneous analysis for the expression of gene, whose expression quantity is extremely small, can be performed.
Then, as a specific application example of the present invention, for example, there exists gene expression analysis using the DNA microarray, and it is possible to increase the speed of its analysis. Specifically, according to the present invention, various kinds of isotopes can be used as labels, and when a stable isotope is used as a label, for example, it is possible to increase the kinds of labels to as many as the number of varieties of stable isotopes (270 kinds). As a result, the amount of information can be significantly increased comparing to a fluorescence method (2 to 6 kinds) that is a conventional labeling method and a radioisotope (approximately 10 kinds).
More specifically, as a label used in a DNA microarray experiment, a probe labeled by nucleotide containing stable isotope such as 39K and 41K, which is the stable isotope of group 1 in the periodic table, 32S and 35S, which is the stable isotope of group 16 in the periodic table, 35Cl and 37Cl, which is the stable isotope of group 17 in the periodic table, or 118Sn and 120Sn, which is the transition metal in the periodic table, for example, is used.
After hybridizing the probe with a target nucleic acid on the DNA microarray, ablation is performed by ultra-short pulse laser, atomic ionization is performed to particles, and then the mass spectrograph detects them, for example, and thus it is possible to determine the quantity of the isotope contained in the hybridized probe. Therefore, the quantity ratio of the probe can be found by calculation.
Herein, the probe has been labeled by fluorochrome in a conventional DNA microarray technique. In the conventional method, approximately 10 minutes were required for detection using exclusive detection equipment after hybridization. However, detection speed can be increased when the present invention is used.
Moreover, only two kinds of fluorochrome (Cy-3, Cy-5) are currently used, and it is not expected to rapidly increase. On the other hand, it is possible to increase the kinds of labels to as many as 270 kinds when the stable isotope is used.
Further, the gene expression data of the DNA microarray is obtained as a relative value to a reference sample. In short, it is difficult to compare the data of the large number of samples between experiments in the conventional DNA microarray experiment where only two kinds of fluorescent label can be used.
However, if a plurality of probes (three kinds or more) labeled by different elements are mixed and simultaneously hybridized with the target, and when the multi-channeled DNA microarray that is measured by the method of analyzing polymer of the present invention, which uses laser ablation is used, data of plural samples can be compared.
Consequently, the present invention is one capable of establishing a highly sensitive and high-speed mass analysis by various kinds of stable isotopic tracer, and therefore, the present invention can be applicable to all fields of research, where labeling is performed by fluorochrome or radioisotope.
Further, according to the present invention, since stable isotope can be used for the labeled element without using radioisotope and no restriction is imposed in facility used in this case, installation in medical facilities and private enterprises is made possible, and its spillover effects is unmeasurable.